• Journal of Applied Biological Chemistry
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• v.64 no.4
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• pp.323-331
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• 2021

Lincomycin is a lincosamide antibiotic isolated from the actinomycete Streptomyces lincolnensis. Moreover, it has been found to be effective against infections caused by Staphylococcus, Streptococcus, and Bacteroides fragillis. To identify the melanin-inducing properties of lincomycin, we used B16F10 melanoma cells in this study. The melanin content and intracellular tyrosinase activity in the cells were increased by lincomycin, without any cytotoxicity. Western blot analysis indicated that the protein expressions of tyrosinase, tyrosinase related protein 1 (TRP1) and TRP2 increased after lincomycin treatment. In addition, lincomycin enhanced the expression of master transcription regulator of melanogenesis, a microphthalmia-associated transcription factor (MITF). Lincomycin also increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and decreased the AKT phosphorylation. Moreover, the activation of tyrosinase activity by lincomycin was inhibited by the treatment with SB203580, which is p38 inhibitor. Furthermore, we also found that lincomycin-induced tyrosinase expression was reduced by H-89, a specific protein kinase A (PKA) inhibitor. These results indicate that lincomycin stimulate melanogenesis via MITF activation via p38 MAPK, AKT, and PKA signal pathways. Thus, lincomycin can potentially be used for treatment of hypopigmentation disorders.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.2
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• pp.113-123
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• 2022

Diarylpropionitrile (DPN), a selective agonist for estrogen receptor β (ERβ), has been reported to regulate various hormonal responses through activation of ERβ in tissues including the mammary gland and brain. However, the effect of DPN on melanogenesis independent of ERβ has not been studied. The aim of this study is to examine the possibility of anti-melanogenic effect of DPN and its underlying mechanism. Melanin contents and cellular tyrosinase activity assay indicated that DPN inhibited melanin biosynthesis in alpha-melanocyte stimulating hormone-stimulated B16F10 melanoma cell line. However, DPN had no direct influence on in vitro tyrosinase catalytic activity. On the other hand, 17β-estradiol had no effect on inhibition of melanogenesis, suggesting that the DPN-mediated suppression of melanin production was not related with estrogen signaling pathway. Immunoblotting analysis showed that DPN down-regulated the expression of microphthalmia-associated transcription factor (MITF), a central transcription factor of melanogenesis and its down-stream genes including tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. Also, DPN attenuated the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). Additionally, DPN suppressed the melanin synthesis in UVB-irradiated HaCaT conditioned media culture system suggesting that DPN has potential as an anti-melanogenic activity in physiological conditions. Collectively, our data show that DPN inhibits melanogenesis via downregulation of PKA/CREB/MITF signaling pathway.

• Molecular & Cellular Toxicology
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• v.3 no.4
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• pp.215-221
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• 2007

Neuronal cell toxicity induced by decreased nitric oxide (NO) production may be caused by modulation of constitutive neuronal NO synthase (nNOS). We used lead acetate ($Pb^{2+}$) to modulate physiological NO release and the related pathways of protein kinases like PKC, CaM-KII, and PKA in CATH.a cells, a dopaminergic cell line that has constitutive nNOS activity. In the cells treated with $Pb^{2+}$, cell viability and modulation (phosphorylation) levels of nNOS were determined by MTT assay and Western blot analysis, respectively. nNOS reductase activity (cytochrome c) was also assessed to compare the phosphorylation site-specific nNOS activity. nNOS activity was also determined by NADPH consumption rates. $Pb^{2+}$ treatment alone increased the phosphorylation of nNOS with decreased reductase activity. The phosphorylation levels increased markedly with decreased nNOS reductase activity, when $Pb^{2+}$ was combined with inhibitors for two (PKC and CaM-KII) or three (PKA, PKC and CaM-KII) protein kinases. Interestingly, when the cells were exposed to $Pb^{2+}$ plus PKC or CaM-KII inhibitor, the nNOS was phosphorylated strongly with the lowest activity. However, the levels of phosphorylated nNOS following $Pb^{2+}$ treatment decreased significantly after combined treatment with the PKA inhibitor, and $Pb^{2+}$-induced suppression of reductase activity did not occur. These results demonstrate that physiological NO release in the neuronal cells exposed to $Pb^{2+}$ can be decreased by PKA-mediated nNOS phosphorylation that may be caused by interactions with PKC and/or CaM-KII.

• Korean Journal of Plant Resources
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• v.35 no.2
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• pp.372-379
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• 2022

This study investigated the effect of lemon balm (Melissa officinalis) extract on improving blood triglycerides according to the extraction solvent using 3T3-L1 cells. Lemon balm was extracted with water (MOW100), 70% ethanol (MOE70), 50% ethanol (MOE50), and 30% ethanol (MOE30). To verify its efficacy on improving blood triglycerides, cell viability, lipid accumulation, triglyceride (TG) content, and expressions of protein kinase A (PKA), adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), perilipin, and CGI-58 proteins were evaluated. Cytotoxicity was not evident up to an extract concentration of 1 mg/mL. Lipid accumulation and TG content were decreased in a concentration-dependent manner compared to their levels in the control group. When the MOW100 extract was applied at a concentration of 0.2 mg/mL, an inhibitory effect was evident, with lipid accumulation inhibited by 21.3% and TG content reduced by 32.7%. PKA phosphorylation and ATGL HSL and CGI-58 levels were increased. The data indicate that lemon balm extract obtained using water is more efficacious than extracted with ethanol. The aqueous extract shows potential in triglyceride control through lipolysis and lowering triglyceride levels.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.8
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• pp.919-928
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• 2017

The immuno-enhancing effects of alginate oligosaccharides from Sargassum coreanum were investigated. The alginate oligosaccharides were produced by an alginate-degrading enzyme from S. oneidensis PKA 1008. The degraded alginate oligosaccharides were visualized by thin-layer chromatography developed using a solvent system of 1-butanol/methanol/water, 4:1:2 (v/v/v). Alginate was degraded into dimmers at 60 h. As a result, the levels of Th1 cytokine [interferon $(IFN)-{\gamma}$ and interleukin (IL)-2] and Th2 cytokine (IL-6 and IL-10) increased with increasing incubation time compared to the control in vitro. Enzymatic extract treatment promoted proliferation of splenocytes at concentrations of 100 and 200 mg/kg at 24 h in vivo. Secretion of $IFN-{\gamma}$ and IL-2 significantly increased in a dose-dependent manner at 24 h as well as induced higher production of IgG2a in serum. Natural killer cell activity was measured and tended to increase. In addition, complete blood cell counts increased in a dose-dependent manner. These results indicate that alginate oligosaccharides produced by crude enzyme from S. oneidensis PKA 1008 may have significant immune activities.

• KSBB Journal
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• v.21 no.6 s.101
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• pp.466-473
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• 2006

Impaired insulin secretion from pancreatic beta-cells in response to glucose is an important feature in the pathology of non-insulin-dependent diabetes mellitus (NIDDM). In the course of screening for useful insulin secretagogues, we have isolated and identified YHB-2017 (Genistein) as a insulin secretion potentiator from fermentation broths of our in-house microbial library. The insulinotropic activity of YHB-2017 in isolated rat pancreatic islets was exerted only at high concentration of glucose (8.3-16 mM) but not at low concentration of glucose (3.3-5.5 mM). Also, in perifusion study with isolated rat pancreatic islets, YHB-2017 stimulated insulin secretion in a time-dependent manner when YHB-2017 was added to KRB buffer containing 16 mM glucose. In the presence of $200\;{\mu}M$ diazoxide and 35 mM KCI, which stimulates maximum $Ca^{2+}$ influx independently of KATP channel, YHB-2017 enhanced KATP channel-independent insulin secretion at high concentration glucose (16 mM). To elucidate the mechanisms of the glucose-dependent potentiation effect of YHB-2017, pharmacologic inhibitors for protein kinase A, protein kinase C and calcium/calmodulin kinase II were pre-treated and then the potentiation effect of YHB-2017 on insulin secretion was investigated. Pre-treatment of H89 as a PKA inhibitor had a significant inhibitory effect on YHB-2017-induced potentiation effect. Furthermore, western immunoblotting analyses revealed that YHB-2017 increased phosphorylation of PKA substrates and cAMP response element-binding protein (CREB) under high concentration of glucose. These results demonstrated that the insulinotropic effect of YHB-2017 is mediated through PKA signal pathway and activated amplifying $K_{ATP}$ channel-independent insulin secretion pathway.

• KSBB Journal
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• v.22 no.5
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• pp.282-287
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• 2007

The antiviral mechanism of KT5720 is known to inhibit the cAMP-dependent protein kinase (PKA), on the VSV infection in BHK-21 cell cultures. The virus inducted CPE (cell rounding) was almost completely suppressed by KT5720 at 5 uM. The inhibitor, however, did not affect the replication of the virus and the synthesis of viral macromolecules. Immunological studies showed the viral matrix (M) protein displayed intimate association with the cytoskeletal components and probably the cell rounding. KT5720, did not block the cytoskeletal disruption, while the cell rounding was suppressed. These observations suggest that the interaction between the viral M protein and the cytoskeletal components may not be enough to cause the morphological change of the cell. And, the KT5720-sensitive function may be involved in developing the VSV-induced CPE, but not essential for the virus replications.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.20 no.3
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• pp.51-62
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• 2007

Objective : The aim of this study is to assess the effect of Schizandrae Fructus on melanin synthesis inhibition and whitening effect. Methods : We assessed inhibitory effects of Schizandrae Fructus on melanin-release from B16F10, on melanin production in B16F10, on mushroom tyrosinase activity in vitro, on tyrosinase activity in B16F10 and effect of Schizandrae Fructus on the expression tyrosinase, TRP-1, TRP-2, PKA, ERK-1 ERK-2, AKT-1, MITF in B16F10. Results and Conclusion : 1. Schizandrae Fructus inhibited melanin-release, melanin production in B16F10. 2. Schizandrae Fructus inhibited tyrosinase activity in vitro and in B16F10. 3. Schizandrae Fructus suppressed the expression of tyrosinase, TRP-1, TRP-2, PKA, ERK-2 in B16F10.

• Bulletin of the Korean Chemical Society
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• v.18 no.4
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• pp.428-432
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• 1997

The dephosphorylation specificity of protein phosphatase 2A (PP2A), calcineurin (PP2B) and protein phosphatase 2C (PP2C) were studied in vitro using myelin basic protein (MBP) as a model substrate which was fully phosphorylated at multiple sites by protein kinase C (PKC) or cyclic AMP-dependent protein kinase (PKA). In order to determine the site specificity of phosphates in myelin basic protein, the protein was digested with trypsin and the radioactive phosphopeptide fragments were isolated by high performance liquid chromatography (HPLC) on reversed-phase column. Subsequent analysis and/or sequential manual Edman degradation of the purified phosphopeptides revealed that Thr-65 and Ser-115 were most extensively phophorylated by PKA and Ser-55 by PKC. For the dephosphorylation kinetics, the phosphorylated MBP was treated with calcineurin or PP2C with various time intervals and the reaction was terminated by direct tryptic digest. Both Thr-65 and Ser-115 residues were dephosphorylated more rapidly than any other ones by phosphatases. However it can be differentiated further by first-order kinetics that the PP2B dephosphorylated both Thr-65 and Ser-115 with almost same manner, whereas PP2C dephosphorylated somewhat preferentially the Ser-115.

• Nuclear Engineering and Technology
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• v.54 no.8
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• pp.2783-2791
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• 2022

Molecular dynamics simulations were performed to predict the behavior of graphite atoms under neutron irradiation using large-scale atomic/molecular massively parallel simulator (LAMMPS) package with adaptive intermolecular reactive empirical bond order (AIREBOM) potential. Defect structures of graphite were compared with results from previous studies by means of density functional theory (DFT) calculations. The quantitative relation between primary knock-on atom (PKA) energy and irradiation damage on graphite was calculated. and the effect of PKA direction on the amount of defects is estimated by counting displaced atoms. Defects are classified into four groups: structural defects, energy defects, vacancies, and near-defect structures, where a structural defect is further subdivided into six types by decision tree method which is one of the supervised machine learning techniques.